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Biology 442 - Human Genetics

Chromosome Analysis: Karyotyping, Banding and FISH

Chromatin refers to "colored" material in the nucleus. We can think
of it as chromosomal material. Heterochromatin refers to non transcriptionally
active chromatin. There is facultative heterochromatin such as the X
chromosome which is capable of being active transcriptionally although
it is not always active and constitutive heterochromatin such as the
q arm of the Y chromosome or the centromeric heterochromatin which is
never transcriptionally active. Euchromatin refers to transcriptionally
active chromatin.

Thenormal or euploid number of chromosomes in human somatic
cells is 46. Cells with 46 chromosomes are said to be diploid and the
abbreviation used is 2n. There are 22 pairs of autosomes and one pair
of sex chromosomes, the X or Y. Females have two X chromosomes and males
have an X and a Y. Gametes contain 23 chromosomes, half the number of
chromosomes found in somatic cells. They are said to be haploid which
is abbreviated n. Fertilization is the fusion of two gametes, a sperm
(n) and an egg (n), to form the zygote which has the diploid number
(2n). Each parent contributes one member of each homologous pair of
chromosomes to the offspring. The egg carries 22 autosomes and an X
chromosome while the sperm has 22 autosomes and either an X or a Y.
Occasionally, there are tetraploid cells (4n) in the liver. Cancer cells
often have an abnormal number of chromosomes (heteroploid) and/or structurally
abnormal chromosomes.

Karyotyping

Karyotyping is the process of preparing chromosomes for analysis. The
tissues used vary but the most common tissues sampled are lymphocytes
(white blood cells), skin fibroblasts, amniotic fluid cells, gonadal
tissue, chorionic villi (CVS) and bone marrow. Direct preparations without
the use of a mitogen are made on bone marrow, lymphocytes, gonadal tissue,
CVS, and tumors. When cultured, cells are placed in a nutrient solution
containing amino acids and other nutrients, a pH indicator, antibiotics,
with or without mitogens (phytohemaglutinin,PHA, or pokeweed mitogen)
and then grown at 37°C in CO2 atmosphere.
Lymphocytes are cultured three days and amniotic cells, fibroblasts,
CVS, and other fetal tissues are cultured ten days. Colcemid, which
inhibits the assembly of microtubules of the spindle, is added prior
to harvest to stop mitosis and to collect sufficient cells with prophase-prometaphase
chromosomes. Cells may be grown on microscope slides in sterile chambers
or they may be attached to the flask in which they are grown. If release
of cells from substrate is necessary, trypsin and EDTA are added. The
cells are then centrifuged to remove the culture medium and/or the trypsin
and EDTA. A hypotonic solution is added to swell the cells followed
by centrifugation and fixation in methanol:acetic acid. The cells are
dropped onto slides if they were not grown on slides. Most banding procedures
require pretreatment before staining. Trypsin and Giemsa produce G-banded
chromosomes (GTG).

PROPERTIES OF CHROMOSOME BANDS SEEN WITH STANDARD GIEMSA STAINING

DARK BANDS (G BANDS)

PALE BANDS (CORRESPOND TO R
BANDS)

Stain strongly with dyes that bind preferentially
to AT-rich regions such as Giemsa and Quinicrine

Stain weakly with Giemsa and Quinicrine

May be comparatively AT-rich

Maybe comparatively GC-rich

DNase insensitive

DNase sensitive

Condense early during the cell cycle but replicate
late

Condense late during cell cycle but replicate early

Gene poor. Genes may be large because exons are
often separated by very large introns

Gene rich, Genes are comparatively small because
of close clustering of exons

LINE rich but may be poor in Alu repeats

LINE poor but may be enriched in Alu repeats

The slides are scanned for metaphase spreads and usually 10 to 30 cells
are analyzed under the microscope by a cytogeneticist. When a good spread
(minimum number of overlapping chromosomes) is found, a photograph is
taken or the analysis is done by a computer. The chromosomes are arranged
in a standard presentation format of longest to shortest. Actually chromosome
21 is smaller than chromosome 22, however, since Trisomy 21 (Down Syndrome)
had already been named, it was decided to leave the numbering system
as it was. In a standard karyotype, the chromosomes are arranged by
size and location of the centromere. There are 7 groups: A, 1 - 3 long
metacentrics; B, 4 - 5 long sub-metacentrics; C, 6 - 12 sub-metacentrics;
D, 13 - 15 acrocentrics which may have satellites (normal chromosome
polymorphisms) on the p arm; E, 16 - 18, sub-metacentrics; F, 19 - 20,
short metacentrics; G, 21 - 22, acrocentrics with or without satellites
(normal polymorphism) on the p arm; the X chromosome is a sub-metacentric
which belongs in the C group and the Y chromosome is a sub-metacentric
which can be placed in the G group on the basis of size. The shorter
arm is placed on top in a karyotype and is called the p arm, the longer
arm is on the bottom and is called the q arm.

1.
Metacentric 2.
Sub metacentric 3.
Acrocentric

There is a standardized way of denoting the bands on banded chromosomes.
The karyotypes of normal individuals are almost identical with the exception
of some chromosome polymorphisms such as the satellites of the D and
G group chromosomes and some heterochromatic regions around the centromeres
of 1, 9, 16 and Yq. Each chromosome arm is numbered beginning with the
centromere: 11 or 11.1 at the centromere, e.g., 21.2 : Region = 2, Band
= 1 and sub band = 0.2

The chromosomes of the great apes are remarkably similar to ours except
for some chromosome rearrangements. Chimps, orangutans and gorillas
have 48 instead of 46. The human chromosome #2 is the result of a translocation
of two chromosomes found in the great apes. There has been a Down Syndrome
chimpanzee with an extra small chromosome homologous to our 21.

Banding techniques

GTG (pretreatment with trypsin then Giemsa stain) or GTW (Giemsa-trypsin-Wrights);
Q (quinicrine, fluorescent bands); R (reverse banding) requires heating
in a saline buffer prior to staining with Giemsa or Quinicrine and is
often called T (telomeric) banding since it also highlights the telomeres;
C (centromere or constitutive heterochromatin) stains the heterochromatin
in the centromeres, especially of chromosomes 1, 9, and 16; NOR (nucleolar
organizing regions) silver nitrate stains selectively the satellite
stalks of the acrocentric chromosomes; sister chromatid exchange (SCE)
requires replication in 5 BUdR, an analog of thymidine followed by Höescht
stain. SCE is used in the diagnosis of Bloom Syndrome, a genetic disorder
that results in chromosome breakage. It is also used as a test for mutagenicity.

GTG banded human chromosomes with banded cartoon along
side

C Banding (Centromere or Constitutive Heterochromatin)

Sister Chromatid Exchange (left is normal; right is
Bloom Syndrome)

The Banding Numbering System

Until the early 1970's the only stains for chromosomes produced "solid
staining". Later with the use of Giemsa stain, G-banding resulted.
In brief, banding patterns/numbers are based solely on G-banded straining
patterns. We now know that dark G bands and bright Q bands are AT rich,
late replicating, gene poor, and contain LINE repeats. G light bands
and dull Q bands are GC rich, early replicating, gene rich, and contain
SINE/Alu sequences.

The original assignment of band numbers were of "regions"
and there were far fewer bands seen at the time, therefore only the
larger "regions" were identified and numbered.....i.e., originally
the numbers referred only to the larger regions of dark and light bands
as seen by Giemsa staining.

The numbering of regions begins at the centromere of each chromosome
arm (p or q) and moves outward so that the larger "regions"
are numbered 1, 2, 3, etc. depending on the number of large dark/light
bands seen. The regions begin and end half way through the band. As
the techniques of chromosome preparation for banding improved, the higher
resolution revealed more (smaller) bands and subbands within the major
regions and so more numbers within the regions were assigned.

The major dark and light areas starting from the centromere each have
a number. Each band is numbered beginning with 11 or 11.1 at the centromere.
When you see numbers assigned, the first number is the region, the second
is the band within the region, and the third number (a decimal) is a
sub-band within the band. For example: 31.2 means 3 is the region, 1
is the band, 0.2 is the sub-band. Regular banding produces about 550
light and dark bands. High resolution banding (HRB) to look for microdeletions
requires the cell culture to be partially synchronized to catch the
cells in pro-metaphase before they condense too much. It produces 800
or more bands. We now have regions, bands, and sub bands, and even sub
subbands, e.g., 12.2 or 11.21. This system makes it easier and more
accurate for scientists to communicate with one another.

Q
banded Cat chromosomes Q
banding FISH
Telomere Cat Chromosomes

Human chromosome characterization nomenclature

46, XX is the normal female karyotype; 46, XY is the normal male karyotype.
X and Y are the sex chromosomes; all others are called autosomes. 47,XY,+21
means there is an extra #21 chromosome (a trisomy); 45,X, means there
is one less chromosome, namely one sex chromosome is missing (a monosomy).
Mosaicism is denoted by a "/", e.g., 45,X/46,XX indicates a Turner female
with two cell lines, one normal and one missing the second X. This arises
from post zygotic non disjunction, after fertilization and in mitosis
of the zygote or embryo.

Haploid = n is the number of pairs of chromosomes. Most organisms are
diploid, 2n, and their gametes are haploid, n. Polyploidy refers to
having multiple sets of chromosomes: 3n (triploid); 4n (tetraploid).
Aneuploidy refers to having (usually) one more or less of the diploid
number of chromosomes: trisomies (one extra); monosomies (one less).

FISH

FISH (fluorescence in situ hybridization) has become a commonly
used staining technique where known sequences of DNA are fluorescent
labeled and hybridized to chromosomes.The process depends on complementary
base pairing, similar to Southern blots. In situ refers to the
fact that the fluorescent DNA probe is hybridized to the DNA of interphase
nuclei or metaphase chromosomes which have been fixed on a slide. FISH
uses locus specific DNA probes, repetitive DNA such as the centromeric
alpha satellite DNA or whole chromosome painting (wcp) or spectral painting
probes. FISH can be used in the identification of marker chromosomes,
rings and other structural anomalies using probes which are specific
for each chromosome (painting probes). Below, you can see examples of
chromosome abnormalities with comparisons of GTG banding and FISH analysis.
There is a picture of a 3p+ where the "+" was shown to come from chromosome
13; an iso 5q which was actually a 5 with a piece of 6; an 8 p+ where
the + was a piece of 21; an 8 p+ where the + was a piece of 22. FISH
is now used almost routinely for microdeletion syndromes which we will
discuss later.

Centromeric repeat probes which bind to the unique satellite sequences
give a large, diffuse but intense signal around the centromere. They
can detect aneuploidies such as trisomies 21, 18, 13 even in interphase
nuclei. They are not useful in detecting structural abnormalities.

Locus specific probes give a sharp, discrete signal at the relevant
positions in each chromatid of a metaphase chromosome; they can be used
to identify or confirm microdeletion (or macrodeletion) syndromes.
An SRY probe can detect the gene responsible for testes formation even
if it has been translocated to another chromosome.

Painting probes can uniformly decorate the entire chromosome
or can be designed to paint the p or q arm, etc. The DNA sequences used
are unique to the chromosome or arm painted. They can be used to identify
the chromosomal origin in structural rearrangements such as translocations,
rings, isochromosomes, etc. The term, "spectral karyotyping" is used
to describe "whole" chromosome specific painting.